The capsular polysaccharide poly(ribosylribitol) phosphate (PRP) of Haemophilus influenzae type b (Hib) is the protective immunogen used in commercially available anti-Hib vaccines. This polysaccharide is a linear polymer of the following repeating unit, ##STR1##
[.fwdarw.3)-.beta.-D-Ribf-(1.fwdarw.1)-D-Ribol-(5-PO.sub.2.fwdarw.], where n is equal to the number of repeat units (RU) (Ribf, ribofuranose; Ribol, ribitol). To improve the immune response of anti-Hib vaccines in the principal recipients, e.g., infants younger than two years old, the polysaccharide has been synthetically coupled to protein carriers to form neoconjugates. See Dick, W. E., and Beurret, M. (1989) Contrib. Microbiol. Immunol. 10, 48-114, and references cited therein. Because of the high average molar mass and wide molar-mass distribution of native PRP (Hennessey, J. P., Jr., Bednar, B.), and (Manam, V. (1993) J. Liq. Chromatogr. 16, 1715-1729), conjugates have been prepared from low molar-mass polysaccharide fragments having narrow, reproducible molar-mass distributions. Such fragments have been generated by controlled hydrolysis, (Egan, W., Schneerson, R., Warner, K., and Zon, G. (1982) J. Am. Chem. Soc. 104, 2898-2910; Zon, G., and Robbins, J. D. (1983) Carbohydr. Res. 114, 103-121), and partial oxidative depolymerization of native polyribosyl ribitol phosphate (PRP). (Anderson, P. W., Pichichero, M. E., Insell, R. A., Betts, R. F., Connuck, D. M., Eby, R., and Smith, D. H. (1986) J. Immunol. 137, 1181-1186).
Molar masses of such polysaccharide preparations have been estimated by size-exclusion chromatography (SEC) using as molar mass standards molecules other than PRP. Because such molecules may behave differently than PRP on an SEC column, this calibration method, termed secondary calibration, introduces the potential for significant errors in the determination of the actual site of the polysaccharide to be analyzed (Hennessey, et al.). The use of secondary calibration introduces the potential for significant errors in the determination of the actual size of the polysaccharide to be analyzed. A series of colorimetric and biochemical assays involving analysis of the concentration of reducing end groups (i.e., reducing end group analysis) on the resulting fragments is another method for estimating the size of polysaccharides. This method however suffers from the problem that the standards used may have different functional groups and react differently in the assays than the actual ends of the polysaccharide to be analyzed, thus giving inaccurate results. See Egan et al. supra and Pillai, S., Ciciriello, S., Koster, M., and Eby, R. (1991) Infect. Immun. 59, 4371-4376. Because such methods measure molar mass indirectly, they suffer from several limitations arising from their reliance upon initial assumptions about the behavior of the PRP fragments relative to standard compounds. Such assumptions have often proven erroneous. (Hennessey et al. supra and Bednar, B., and Hennessey, J. P., Jr. (1993) Carbohydr. Res. 243, 115-130), demonstrated that molar-mass estimation of native PRP and pneumococcal capsular polysaccharides by secondary calibration was not appropriate because of differences in hydrodynamic properties of the secondary standard and the polysaccharide under study. Disadvantages of chemical estimation of molar masses include the tedium of running several assays on the same sample; the necessity of having to rely on assumptions about the uniformity of fragment end groups; and the compounding of error from the mathematical combination of data from the assays.
Another potential error in the reducing-end-group analysis of fragments prepared by partial oxidative depolymerization of PRP (Anderson et al., supra) is the inappropriate use of ribose as a standard. Reducing-end-group analysis is typically done using the Park-Johnson assay (Park, J. T., and Johnson, M. J. (1949) J. Biol. Chem. 282, 149-151). Oxidative cleavage of ribitol results in fragments having dissimilar end groups which have different reactivities in the Park-Johnson assay, even though both terminal end groups possess a terminal O-formylmethyl group. (Groups 1 and 2, below). (Seid, R. C., Boykins, R. A., Liu, D. F., Kimbrough, K. W., Hsieh, C. L., and Eby, R. (1989) Glycoconjugate J. 6, 489-498). ##STR2##
The Park-Johnson assay for detecting reducing end groups in carbohydrates was the only method reported to be used to determine the number average chain length L.sub.n the average number of repeat units per polysaccharide chain) of PRP fragments obtained by oxidative cleavage and used in the vaccines described in Anderson, et al., U.S. Pat. No. 4,902,506. Because ribose was used as the standard for end-group analysis of these fragments, the Park-Johnson assay may have overestimated the average chain length of the fragments by at least 50%, compared to more accurate methods, such as laser-light scattering (LLS), according to data presented below. The data presented below demonstrated that reliance on a single, indirect, colorimetric method for the estimation of molar mass may give erroneous results. Thus, a need exists for precise and consistent methods useful for characterization of vaccines for human recipients.